Monthly Archives: September 2014

During my research placement at the Medical Research Centre for Developmental Neurobiology with Dr Tara Keck, I was able to take part in various experiments and techniques from electrophysiology to measure the potential difference and current of neurons, using computer software ‘Fiji’ to analyse neurons that are Green Fluorescent Protein (GFP) positive in inhibitory cells and immunohistochemistry to identify specific proteins.

My last two days involved carrying out immunohistochemistry to detect Glial Fibrillary Acidic Protein (GFAP). This protein is usually used as a cell marker and is expressed in one of the two types of brain cells called neuroglia, particularly astrocytes.

The image below displays astrocytes that express GFAP in the subventricular zone. This region of the brain is recognized as being the site of neurogenesis, the growth of new neurons which can also be seen in the image as the collection of cells on the left hand side of the subventricular zone.

My two week experience led me to the discovery of what to expect in neuroscience research and the rewarding feeling of working alongside post doctors and PhD students. The work can become somewhat frustrating when the day does not go as planned however the images and results produced are very pleasing.

The research my laboratory focused on was finding important proteins or eliminating genes that could affect neurodegeneration. One particular disease which is focused on is dentatorubral-pallidoluysian atrophy (DRPLA). The mating scheme diagram you can see in the image below is a hypothetical one created as a practice for designing ones in the lab. My supervisor Catarina regularly designed the mating schemes for flies so that she would be able to generate flies with certain genotypes to observe. It was amazing to find that some of the diagrams she had to make involved crossing the different stocks of flies 11 times, just to get the desired genotype.

Morgan describes her time in the MRC Centre for Developmental Neurobiology at King’s College London.

Things were more than busy on my first Wednesday whilst working at the lab for my work placement at the MRC Centre for Developmental Neurobiology at King’s College, not only was it my turn to work my way through the protocol for preparing coffee for the department that morning, but it was dissection day for Ivana (my postdoctoral supervisor). That meant that I had the pleasure in assisting her with the oculomotor nerve dissection she had planned for that day. As part of her project she needed to isolate cranial motor neurons from the this nerve found in the brain and later grow these cells in a culture to observe how their axons are guided to their targets during development.

Thursday proved to be busier in the lab as first thing that morning we got stuck into electroporation. Electroporation is a method that uses an externally applied electrical field on the cells of interest to increase the electrical conductivity and permeability of the cell plasma membrane. It is a way of introducing a substance into a cell, here we used it to insert DNA into our chosen cell (a process known as transfection).

The second week of my neuroscience placement has seen me come into contact with some pioneering methods used within genetic science and many of the biosciences. One of which is the use of the polymerase chain reaction (PCR). This is a form of genotyping which allowed us to determine the differences in genotype of the individual organisms we were studying by examining their DNA sequences and comparing them. It involves artificial DNA replication so that samples of DNA can be amplified to generate millions of copies for genetic profiling.

Gel electrophoresis. Now this is cool stuff. This particular technique is a modern molecular biology method used to separate out DNA fragments based on their size so that these fragments can be used for identification and analysis – this can tell us which genes are present in a sample and whether the individual expresses this gene or not. It uses an agarose gel with wells cut into it to place the DNA samples into, this is covered in a buffer solution in an electrophoresis tank with electrodes attached at each end so that a current is passed through attracting the negatively charged DNA and separating it through the gel by base length.

Continued from her previous post, Lucky tells us about her experience with using a fundamental technique in molecular biology – the polymerase chain reaction (PCR)…

During my placement I had the opportunity to observe the isolation of RNA and the process of reverse transcription to get cDNA; with this I learnt how enzymes, for example reverse transcriptase, can be used by scientists to achieve a desired outcome. I was able to take part in PCR where a small sample of DNA is amplified in three main steps: denaturation, annealing and elongation. The samples, including my own, were run through a gel and imaged. My sample appeared as a very bold band which is good.

Over the two weeks spent in the laboratory I have learnt about how significant understanding and testing pathogens (particularly Candida albicans and Candida galbrata) is to preventing human fungal diseases being contracted. I spent time investigating the different strains of yeast at both a molecular level through polymerase chain reactions (PCR) which analysed the yeast strains DNA (see photo A) and therefore genetics alongside microscope studies and also visually by comparing phenotypes which Manduca sexta caterpillars (see photo B) expressed when different strains or dilutions were injected, monitoring them for changes in weight and colour.

Photo A: Agarose gel electrophoresis to analyse PCR products

Photo B: Manduca sexta

I had the opportunity to collect my own data which enabled me to see, from the beginning to the end of the life cycle how powerful fungal pathogens can be and relate that to the importance of all the studies within the lab to preventing illnesses. The members of the lab were varied, including PhD researchers and master students, all of whom have enlightened me as to the broad range of topics science has to offer and the overlap of many different aspects, whilst also explaining different pathways into their scientific careers which has shown me the vast amount of ways to work within the field and that there are opportunities to move on and complete different studies throughout your whole career.

Participating in this placement scheme, alongside Stephanie and other lab members I have learnt a huge amount in just two week and realised that the phrase ‘you learn something new every day’ really is true when working in a scientific career, with around 80% of experiments not actually working scientists have to be patient in order to achieve the end result which could one day save lives.